High Speed super-resolution confocal laser scanning microscope for sub-diffraction analysis at the multi-user Leicester Advanced Imaging Facility

分子、细胞、组织和整个生物体的光学成像为我们解决生物学问题提供了宝贵的信息。如果我们有兴趣了解细胞/组织中的微观事件，以及它们的数量和动态行为，这将有助于我们理解它们的生物学作用，那么这就是字面上的“眼见为实”。例如，如果在细胞膜表面发现它们，它们的作用可能与细胞膜功能有关。如果在细胞的同一位置发现两种蛋白质，它们可能会共同发挥细胞作用。如果这些蛋白质在疾病细胞/组织中缺失，恢复这些蛋白质可能是一种有效的治疗策略。为了突出显示感兴趣的分子的位置，我们需要“标记”分子。与目标分子相关的荧光标记彻底改变了我们研究细胞和组织发育和生理过程的能力。它们既可用于固定样品，也可用于活细胞。这些标记由光学显微镜检测，其中一种称为共聚焦激光扫描显微镜（CLSM）的专用系统起着至关重要的作用。clsm允许我们仅从焦平面收集信号，使用检测器前面的小光圈排除失焦光。因此，我们可以观察细胞和组织内部，而不需要将它们切成几段，也可以观察漂浮在溶液中的特定分子，而不会被周围多余的其他分子所掩盖。clsm可以通过调节像素设置或放大单元格的较小区域来灵活地更改图像的像素分辨率。这意味着大的组织样本和微小的细菌样本都可以通过CLSMs进行分析。图像的质量取决于它们的“分辨率”，它被定义为样本中两点之间的最小距离，这两点仍然可以被检测器（我们的眼睛或相机）区分为单独的点。传统荧光显微镜的分辨率被限制在200纳米左右，这被称为“衍射极限”。在过去的二十年中，新的方法带来了分辨率的实质性改进，根据方法的不同，分辨率可以达到50-120纳米。这一细胞生物学的重大突破被授予2014年诺贝尔化学奖，以表彰“超分辨荧光显微镜的发展”。这些显微镜可以被称为超分辨率显微镜（或纳米显微镜，因为它们提供纳米分辨率）。超分辨率显微镜提供的信息是独一无二的，不能通过任何其他手段获得。例如，电子显微镜（EM）的高分辨率成像，其分辨率现在可以降低到几Ånstrongs，不能取代超分辨率显微镜，因为EM不能使用荧光标记，并且替代方法来定位与观察结构相关的感兴趣的蛋白质是有限的。研究人员意识到了这个问题，并积极致力于此，但用专门的标签精确“标记”感兴趣的蛋白质的方法开发仍在进行中。因此，诸如两种蛋白质是否在特定的细胞结构中共定位之类的问题，需要通过超分辨率显微镜来解决。不出所料，超分辨率显微镜在细胞生物学研究领域迅速变得非常流行。分辨率提高1.7到2倍（约100-120纳米）已成为细胞生物学的新标准。具有超分辨率功能的clsm在商业上是可用的。这些超分辨率clsm可以配备一个探测器，以提高灵敏度和速度，允许对目标分子可能进行动态运动的活细胞进行成像。通过在多用户莱斯特高级成像设施安装这些超分辨率clsm之一，我们的目标是促进世界一流的细胞生物学。

Optical imaging of molecules, cells, tissues and whole organisms brings invaluable information when we address biological questions. It is literally "seeing is believing", if we are interested in finding out about microscopic events in a cell/tissue as well as their quantities and dynamic behaviour that will help us to understand their biological roles. For example, if they are found on the cell surface membrane, their role may be relevant to cell membrane function. If two proteins are found at the same site in a cell, they may work together to deliver a cellular role. If these proteins are missing in disease cells/tissues, restoration of these proteins may be an effective therapeutic strategy.To highlight the locations of the molecules of interest, we need to "mark" the molecules. Fluorescent markers that associate with the target molecules have revolutionized our ability to study cellular and tissue developmental and physiological processes. They can be used in both fixed samples and in live-cells. These markers are detected by optical microscopes, where a specialised system called Confocal Laser Scanning Microscope (CLSM) has been playing a vital role. CLSMs allow us to collect signals only from the focal plane, excluding out-of-focus light using a small aperture in front of the detector. Therefore, we can look inside cells and tissues without physically cutting them into sections and look at specific molecules floating in solution without getting masked by surrounding excess of other molecules. CLSMs have a flexibility to change the pixel resolution of the image by modulating the pixel setting or zooming into a smaller area of the cell. This means that both large tissue samples and tiny bacterial samples can be analysed by CLSMs.The quality of images are dependent on their "resolution", which is defined as the minimal distance between two points in the sample that can still be distinguished by the detector (our eyes or a camera) as separate points. Resolution of conventional fluorescence microscopy is limited to about 200 nm, which is termed the "diffraction limit". In the last two decades, novel methodologies have brought substantial improvements to the resolution, which can become 50-120 nm, dependent on methodologies. This major breakthrough in cell biology was awarded the Nobel Prize in Chemistry in 2014 "for the development of super-resolved fluorescence microscopy". These microscopes can be called super resolution microscopes (or nanoscopes as they offer nanometre resolution). The information provided by super-resolution microscopy is unique and cannot be obtained by any other means. For example, high resolution imaging by electron microscopy (EM), where resolution can be now down to a few Ånstrongs cannot replace super-resolution microscopy as EM cannot use fluorescent markers and alternative methodologies to localise a protein of interest in relation to the observed structures are limited. Researchers are aware of this issue and actively working on it but methodology development to precisely "mark" proteins of interest with specialised tags is still ongoing. Therefore, a question such as whether protein two proteins are co-localising at a particular cellular structure or not, needs to be addressed by super-resolution microscopy. Unsurprisingly, super-resolution microscopy has quickly become very popular in the cell biology research field. An improved resolution by a factor of 1.7 to 2 (about 100-120 nm) has become the new standard in cell biology. CLSMs with super-resolution capability are commercially available. These super-resolution CLSMs can come with a detector for improved sensitivity and speed, allowing imaging of live cells where the target molecules may make dynamic movement. By installing one of these super-resolution CLSMs at the multi-user Leicester Advanced Imaging Facility, we aim to promote world-class cell biology.